Journal: BMC Neuroscience

Article Title: Chronic exposure to high fat diet triggers myelin disruption and interleukin-33 upregulation in hypothalamus

doi: 10.1186/s12868-019-0516-6

Figure Lengend Snippet: List of antibodies in the study for immunofluorescence or western blot analysis

Article Snippet: Polyclonal goat anti-IL-33 , R and D Systems Cat#AF3626, RRID:AB_884269 , Escherichia coli -derived recombinant mouse IL-33 , 1:200 (IF).

Techniques: Immunofluorescence, Western Blot, Purification, Affinity Chromatography, Recombinant, Derivative Assay

Journal:

Article Title: Anti-inflammatory effects of a new tumour necrosis factor-alpha (TNF-?) inhibitor (CNI-1493) in collagen-induced arthritis (CIA) in rats

doi: 10.1046/j.1365-2249.1999.00750.x

Figure Lengend Snippet: Immunostaining of synovial tissue in CNI-1493-treated and non-treated DA rats with CIA. Immunostaining of TNF-α (A,C) and MHC II (B,D) in knee joint tissue from rats with CIA. Synovitis, articular cartilage and bone (dark blue staining) are evident in all figures. (A,B) Stainings in consecutive sections from an animal treated daily with CNI-1493 from start of immunization (rat V in Table 3). (C,D) Consecutive sections in tissue from a placebo-treated animal (rat I in Table 3). Both animals were killed on day 21 post-immunization (p.i.), when maximal signs of inflammation occurred in the placebo-treated group of animals. A massive infiltration of MHC II+ macrophages was recorded in the synovitis of both the CNI-treated (B) and the placebo-treated (D) rat, Yet TNF-α-expressing cells were only abundant in the placebo-treated animal (C), while the number of these cells was dramatically reduced in the CNI-treated rat (A).

Article Snippet: After additional thorough washes in BSS–saponin, sections were incubated overnight at room temperature in a humidified chamber with 50 μl of cytokine-specific antigen affinity-purified antibody (either polyclonal rabbit anti-rat TNF-α (lot no. 8-14; Dr P. van der Meide, Biomedical Primate Research Centre, Rijswijk, The Netherlands), or polyclonal antigen affinity-purified goat anti-rat TNF-α (AF-510; R&D Systems, Minneapolis, MN; used at 2 μg/ml).

Techniques: Immunostaining, Staining, Expressing

Journal: Redox Biology

Article Title: A role for peroxiredoxins in H 2 O 2 - and MEKK-dependent activation of the p38 signaling pathway

doi: 10.1016/j.redox.2019.101340

Figure Lengend Snippet: Mammalian MEKK4 is redox-sensitive and interacts with cytosolic peroxiredoxin Prx2 in response to H 2 O 2 . ( A, A ′) A tagged version of Prx2 (Prx2-SBP) was expressed in HEK293T cells. Cells were treated with H 2 O 2 (100 μM) and lysed at indicated time points. Prx2-SBP was affinity-purified using streptavidin beads. Precipitates ( A ) and whole cell lysates (WCL) ( A ′) were analyzed by SDS-PAGE under reducing (R) and non-reducing (NR) conditions followed by immunoblotting (IB). The immunoblots are representative of 3 independent experiments (n = 3). ( B, B′ ) Complementary affinity purification experiment: A tagged version of MEKK4 (MEKK4-SBP) was expressed in HEK293T cells. Cells were treated with H 2 O 2 (100 μM) and lysed at indicated time points. MEKK4-SBP was affinity-purified using streptavidin beads. Precipitates ( B ) and WCL ( B ′) were analyzed by SDS-PAGE under R and NR conditions followed by IB. e.v.: empty vector, PD: pull down, *: endogenous protein, X: unknown protein. The immunoblots are representative of 3 independent experiments (n = 3).

Article Snippet: Jafrac1 was detected with polyclonal goat anti-Prx2 antibody (R&D Systems #AF3489), Drosophila α -tubulin with a monoclonal mouse antibody (Hybridoma Bank AA4.3), Drosophila p-p38 (Thr180/Tyr182) with a monoclonal rabbit antibody (Cell Signaling Technology #4631), Drosophila p38 with a goat polyclonal antibody (Santa Cruz Biotechnology sc-15714) diluted 1:300, myc-tag with a rabbit monoclonal antibody (Cell Signaling Technology #2278), SBP-tag with a mouse monoclonal antibody (Santa Cruz Biotechnology sc-101595), human Prx2 with a rabbit monoclonal antibody (abcam #109367), MEKK4 with a mouse monoclonal antibody (sc-166196), human p-p38 with a mouse monoclonal antibody (BD #6122889) diluted 1:2000, human p38 with a rabbit polyclonal antibody (Cell Signaling Technology #9212) and human β-tubulin with a rabbit monoclonal antibody (Cell Signaling Technology #2128).

Techniques: Affinity Purification, SDS Page, Western Blot, Plasmid Preparation

Journal: eLife

Article Title: A new family of cell surface located purine transporters in Microsporidia and related fungal endoparasites

doi: 10.7554/eLife.47037

Figure Lengend Snippet:

Article Snippet: Antibody , anti-ThMFS4 peptides (affinity purified, rabbit polyclonal) , BioGenes GmbH (Germany). This study , Peptides: CVKRTNSSNR NVGTAK and CKPEAVLFKR KISLKD , IFA: 1:2, 1:10, or 1:50; WB: 1:1000.

Techniques: Clone Assay, Infection, Virus, Expressing, Recombinant, PCR Cloning, Affinity Purification

Journal: Cancer chemotherapy and pharmacology

Article Title: Characterization of the metabolism of benzaldehyde dimethane sulfonate (NSC 281612, DMS612)

doi: 10.1007/s00280-015-2828-2

Figure Lengend Snippet: Western blot of ALDH1A1 from human RBCs. Lanes from left: lane 1 – ladder (49.9kDa), lanes 2 and 3 - blank, lanes 4 and 5 - 80 μg protein, 6 and 7 - 40 μg protein, 8 and 9 - 20 μg protein, lanes10 and 11 - 10 μg protein, and lane 12 ladder (49.9 kDa).

Article Snippet: Membranes were blocked with 5% nonfat milk in Tris-buffered saline and then probed with primary antibodies ALDH1A1 purified MaxPab rabbit polyclonal antibody (D01P; Abnova, Taipei, Taiwan) overnight at 4 °C.

Techniques: Western Blot

Journal: Journal of Biological Chemistry

Article Title: A Death Receptor-associated Anti-apoptotic Protein, BRE, Inhibits Mitochondrial Apoptotic Pathway

doi: 10.1074/jbc.m408678200

Figure Lengend Snippet: FIG. 4. BRE associates with TNF-R1 and Fas. A, co-immunoprecipitation of TNF-R1 with GS-BRE and V5-BRE using anti-V5 monoclonal antibody from HeLa transiently transfected with the respec- tive expression constructs. MAB2, a third party mouse monoclonal antibody against phosphorylcholine, is the negative control. Immunoblotting was performed using a rabbit anti-TNF-R1 polyclonal antibody (Calbiochem). B, co-immunoprecipitation of GS-BRE (52 kDa) with TNF-R1 and Fas using mouse anti-TNF-R1 monoclonal antibody (clone 16803, R & D Systems) and rabbit anti-Fas polyclonal antibodies (C20, Santa Cruz Biotechnology), respectively. MAB2, rat anti-TGF monoclonal antibody (clone A75-2.1, Pharmingen), and normal rabbit serum are negative controls. GS- BRE was immunoblotted by HRP-conju- gated anti-V5 monoclonal antibody. C, co- immunoprecipitation of Fas with GS-BRE detected by rabbit anti-Fas polyclonal an- tibody (C20). Two monoclonal anti-Fas antibodies (clone DX2, Pharmingen, and clone 13, Transduction Laboratories) are positive controls for Fas immunoprecipi- tation. D, co-precipitation of GS-BRE with Fas immunoprecipitated by 2 anti- Fas antibodies, DX2 and C20. GS-BRE was immunoblotted by HRP-conjugated anti-V5 monoclonal antibody. IP, immu- noprecipitation; WB, Western blotting analysis.

Article Snippet: Reagents—Antibodies (clone or code number) and reagents were purchased from the following sources: rabbit polyclonal anti-TNF-R1 and anti-ubiquitin antibodies (Calbiochem); rabbit polyclonal anti-Fas (C-20), anti-TRADD (H-278), and mouse monoclonal anti-SUMO-1 (D11) antibodies (Santa Cruz Biotechnology, Santa Cruz, CA); mouse monoclonal anti-GAPDH (9.B.88) and anti- -actin (2A2.1) antibodies (U. S. Biological, Swampscott, MA); mouse monoclonal anti-Fas (13), anti-G28, and anti-BiP antibodies (Transduction Laboratories); mouse monoclonal anti-Fas (DX2), anti-cytochrome c (7H8.2C12), rat monoclonal anti-TGF (A75-2.1), rabbit polyclonal anti-BID, and anticaspase-8 (poly-1326) antibodies (Pharmingen); rabbit polyclonal anticleaved caspase-3, anti-cleaved caspase-9, anti-PARP, and mouse monoclonal anti-caspase-8 (1C12) antibodies (Cell Signaling Technology, Beverly, MA); mouse monoclonal anti-Fas IgM antibody (CH11), anti-Smac/DIABLO (78-1-118), and anti-histone H1 (AE-4) antibodies (Upstate Biotechnology, Inc.); rabbit polyclonal anti-pan-phosphoprotein and mouse monoclonal anti- -tubulin (2-28-33) antibodies (Zymed Laboratories Inc.); rabbit polyclonal anti-catalase antibody (Abcam Ltd., Cambridge, UK); mouse monoclonal anti-prohibitin (Ab-1) antibody (Lab Vision Corp., Fremont, CA); mouse monoclonal anti-TNF-R1 antibodies (16803) with and without FITC conjugation, and goat antiTNF-R1 affinity-purified polyclonal antibody (R&D Systems, Minneapolis, MN); mouse monoclonal anti-V5 antibody and the horseradish peroxidase (HRP) conjugate (Invitrogen); HRP-conjugated anti-rabbit Ig secondary antibody (Promega, Madison, WI); FITC-conjugated antirabbit, anti-mouse, HRP-conjugated anti-mouse Ig secondary antibodies, and protease inhibitors mixture (Sigma); human recombinant TNF- (Invitrogen); -protein phosphatase (New England Biolabs, Beverly, MA); GS-BRE (ResGen GeneStorm® Clone for accession number L38616), GS alone expression vector, G418, Zeocin, and Lipofectamine 2000 (Invitrogen).

Techniques: Immunoprecipitation, Transfection, Expressing, Construct, Negative Control, Western Blot, Transduction

Journal: Journal of Biological Chemistry

Article Title: A Death Receptor-associated Anti-apoptotic Protein, BRE, Inhibits Mitochondrial Apoptotic Pathway

doi: 10.1074/jbc.m408678200

Figure Lengend Snippet: FIG. 6. BRE dissociates from TNF-R1, but not from Fas, upon receptor ligation. NHGS2 was treated with 100 ng/ml TNF- (A) or 200 ng/ml CH11 (B). At the indicated time points, cell lysates of A were subjected to immunoprecipitation (IP) by mouse monoclonal anti- TNF-R1 antibody (clone 16803, R&D Systems), and the precipitates were immunoblotted with anti-V5-HRP, rabbit anti-TRADD, and TNF-R1 polyclonal antibodies. The same result was obtained by using a goat anti-TNF-R1 affinity-purified polyclonal antibody (R&D Sys- tems) for immunoprecipitation. Cell lysates of B were subjected to immunoprecipitation by monoclonal anti-Fas (DX2) antibody, and the precipitates were immunoblotted with anti-V5-HRP, anti-caspase-8, and Fas (C20) antibodies. Immunoprecipitation by anti-GAPDH anti- body is the negative control. All the cell lysates were also immuno- blotted directly with anti-V5-HRP for GS-BRE to ensure similar amount of starting proteins for immunoprecipitation and to show no degradation of GS-BRE during the time course study.

Article Snippet: Reagents—Antibodies (clone or code number) and reagents were purchased from the following sources: rabbit polyclonal anti-TNF-R1 and anti-ubiquitin antibodies (Calbiochem); rabbit polyclonal anti-Fas (C-20), anti-TRADD (H-278), and mouse monoclonal anti-SUMO-1 (D11) antibodies (Santa Cruz Biotechnology, Santa Cruz, CA); mouse monoclonal anti-GAPDH (9.B.88) and anti- -actin (2A2.1) antibodies (U. S. Biological, Swampscott, MA); mouse monoclonal anti-Fas (13), anti-G28, and anti-BiP antibodies (Transduction Laboratories); mouse monoclonal anti-Fas (DX2), anti-cytochrome c (7H8.2C12), rat monoclonal anti-TGF (A75-2.1), rabbit polyclonal anti-BID, and anticaspase-8 (poly-1326) antibodies (Pharmingen); rabbit polyclonal anticleaved caspase-3, anti-cleaved caspase-9, anti-PARP, and mouse monoclonal anti-caspase-8 (1C12) antibodies (Cell Signaling Technology, Beverly, MA); mouse monoclonal anti-Fas IgM antibody (CH11), anti-Smac/DIABLO (78-1-118), and anti-histone H1 (AE-4) antibodies (Upstate Biotechnology, Inc.); rabbit polyclonal anti-pan-phosphoprotein and mouse monoclonal anti- -tubulin (2-28-33) antibodies (Zymed Laboratories Inc.); rabbit polyclonal anti-catalase antibody (Abcam Ltd., Cambridge, UK); mouse monoclonal anti-prohibitin (Ab-1) antibody (Lab Vision Corp., Fremont, CA); mouse monoclonal anti-TNF-R1 antibodies (16803) with and without FITC conjugation, and goat antiTNF-R1 affinity-purified polyclonal antibody (R&D Systems, Minneapolis, MN); mouse monoclonal anti-V5 antibody and the horseradish peroxidase (HRP) conjugate (Invitrogen); HRP-conjugated anti-rabbit Ig secondary antibody (Promega, Madison, WI); FITC-conjugated antirabbit, anti-mouse, HRP-conjugated anti-mouse Ig secondary antibodies, and protease inhibitors mixture (Sigma); human recombinant TNF- (Invitrogen); -protein phosphatase (New England Biolabs, Beverly, MA); GS-BRE (ResGen GeneStorm® Clone for accession number L38616), GS alone expression vector, G418, Zeocin, and Lipofectamine 2000 (Invitrogen).

Techniques: Ligation, Immunoprecipitation, Affinity Purification, Negative Control

Journal: Journal of Biological Chemistry

Article Title: Detrimental Role for Human High Temperature Requirement Serine Protease A1 (HTRA1) in the Pathogenesis of Intervertebral Disc (IVD) Degeneration

doi: 10.1074/jbc.m112.341032

Figure Lengend Snippet: FIGURE 4. Stimulation of IVD cells with HTRA1-generated fibronectin fragments. A, concentrated protein supernatants (15 g) from IVD cells treated for 24 h without or with HTRA1mac (5 g/ml) or HTRA1macSA (5 g/ml) were subjected to immunoblotting using antibody Mab1935 specific for the fibronectincarboxyl-terminalheparin-bindingdomain(Cterminus)orMab1936specificforthefibronectinamino-terminalfibrin-andheparin-bindingdomain (N terminus). Fibronectin fragments containing the amino-terminal fibrin- and heparin-binding domain are identified by the closed arrowhead. B, purified human plasma-derived fibronectin (Fn) was incubated with HTRA1mac or HTRA1macSA at equimolar concentrations in TBS, pH 8.5, for 16 h at 37 °C, and samples were loaded onto a 4–15% gradient gel and stained with Coomassie Blue. Fibronectin and recombinant HTRA1 alone were also loaded and served as controls. C, an equimolar concentration of human plasma-derived fibronectin and HTRA1mac were incubated for 16 h, and fibronectin fragments were visualized by Western blot analysis using the antibodies described in A. D, equimolar concentrations of fibronectin (20 g) and HTRA1mac (5 g) were incubated for 16 h, and fibronectin fragments were purified by affinity chromatography. IVD cells were incubated with purified HTRA1-digested fibronectin (FnHTRA1mac) for 24 h, and expression levels of MMP1, MMP3, and MMP13 mRNA were determined by qRT-PCR and the -fold change as compared with untreatedcontrolswasdeterminedusingthe2CTmethod.Additionalcultureswereincubatedwitheitheraffinity-purifiedTris-bufferedsaline,pH7.6(TBS), fibronectin (Fn), or HTRA1 (HTRA1mac) or left untreated (Control). Data are representative of two separate experiments performed using IVD cells from two patients. Shown are results of triplicate determinations S.D. *, p 0.01, as determined by one-way ANOVA.

Article Snippet: Materials—Human fibronectin and rabbit IgG were purchased from R & D Systems (Abingdon, UK).

Techniques: Generated, Western Blot, Binding Assay, Purification, Clinical Proteomics, Derivative Assay, Incubation, Staining, Recombinant, Concentration Assay, Affinity Chromatography, Expressing, Quantitative RT-PCR, Control

Journal: Journal of Biological Chemistry

Article Title: Detrimental Role for Human High Temperature Requirement Serine Protease A1 (HTRA1) in the Pathogenesis of Intervertebral Disc (IVD) Degeneration

doi: 10.1074/jbc.m112.341032

Figure Lengend Snippet: FIGURE 5. Detection of fibronectin fragments in degenerated IVD tissue. A, fibronectin (FN) mRNA levels in intact IVD tissue samples from patients (n 36) with varying degrees of IVD degeneration were determined by qRT-PCR and presented as 2CT S.E. (error bars). B, correlation study between FN and HTRA1 mRNA levels (2CT) in patient IVD tissue samples (n 36). R2, square of correlation coefficient; p 0.01 as determined from Pearson’s correlation coefficient. C, protein extracts from patient IVD tissues (n 12) were loaded onto a 12% SDS-polyacrylamide gel, and immunoblotting was performed using a monoclonal antibody (Mab1936) specific for the amino-terminal fibrin- and heparin-binding domain. D, the PVDF membrane used in C was stained with Coomassie Blue in order to confirm equal protein loading. Lane 1, HTRA1-digested human plasma-derived fibronectin; lanes 2–4, non-degenerated (ND) discs; lanes 5–7, mildly degenerated discs; lanes 8–10, moderately degenerated discs; lanes 11–13, severely degenerated discs.

Article Snippet: Materials—Human fibronectin and rabbit IgG were purchased from R & D Systems (Abingdon, UK).

Techniques: Quantitative RT-PCR, Western Blot, Binding Assay, Membrane, Staining, Clinical Proteomics, Derivative Assay

Journal: Journal of Biological Chemistry

Article Title: Detrimental Role for Human High Temperature Requirement Serine Protease A1 (HTRA1) in the Pathogenesis of Intervertebral Disc (IVD) Degeneration

doi: 10.1074/jbc.m112.341032

Figure Lengend Snippet: FIGURE 6. A theoretical model for the role of HTRA1 in IVD degeneration. Based on our findings, we propose that HTRA1 accumulates in IVD tissue undergoing degeneration and stimulates MMP production by resident cells in a predominantly protease-dependent manner, via activation of the MEK pathway. Furthermore, we suggest that the stimulatory effects of HTRA1 on IVD cells are mediated indirectly through its ability to generate fibronectin fragments, although other routes of cellular activation cannot be ruled out. IDD, intervertebral disc degeneration.

Article Snippet: Materials—Human fibronectin and rabbit IgG were purchased from R & D Systems (Abingdon, UK).

Techniques: Activation Assay